Construction Of Nanopore Single Molecular Sensor And Its Preliminary Evaluation

As a low-cost,high sensitivity and high throughput single molecule sensing system,the nanopore sensor has received more and more attention in the past twenty years.At present,the commonly used nanopores are still biological nanopores.Due to its good repeatability and stability,and higher detection accuracy,biological nanopores was widely applied on various organic and inorganic molecular detection and analysis,as well as study of their reaction mechanism at single molecular level.Most commonly used biological nanopore are alpha hemolysin??-HL?protein and mycobacterium smegmatis porin?MspA?.However,the wild type proteins can't directly be used for nanopore analysis because their spontaneous blockage and faster translocation rate.Mutant proteins can be formed by using some protein engineering technology,which can finally achieve specificity for the identification of the target analytes,lower the translocation speed,and to improve the detection accuracy.From this point,we firstly constructed the single molecule detection system based on biological nanopores,and studied the factors which could affect its stability.In addition,the alpha hemolysin nanopore was utilized to study the unzipping kinetic of duplex DNA,and finally achieved discrimination of single nucleotide differences between duplex DNA structures.Protein engineering technology was used to form a novel MspA protein?Mut-MspA?mutation based on wild type MspA protein,which was then applied to detect and discriminate four types of DNA homopolymer,and finally used on construction of higher resolution nanopore detection system.The contents of this thesis including:1.Construction of biological nanopore system for single molecule detectionIn this work,a cross-scale method of fabricating nano-electrodes was proposed,and a 10 nm-width nano-electrode was got from 5?m-width aurum?Au?line.Fabrication procedures involves four steps:?i?Depositing of Au-Si₃N₄ structure by Low Pressure Chemical Vapor Deposition?LPCVD?and Electron Beam Evaporation,?ii?Etching a suitable width of nano-electrode by microscopes,?iii?Depositing 100nm thickness Si₃N₄ on the Au-Si₃N₄ by LPCVD,?iv?Drilling a suitable diameter hole on the sandwich structure by Helium-ion microscopes?HIM?.Compared with the traditional methods,only electron microscopes were involved in this process,it was a simple and effective method to fabricate electrode-embedded nanopore.The feasibility of the method has been demonstrated by practice and a 10 nm nanopore with 10 nm-width nano-electrodes was fabricated by this method.This method will be a new way to fabricate transverse electrode-embedded nanopore in fabricating smaller than 2 nm gap of nano-electrodes in the future.2.Construction of biological nanopore system for single molecule detectionWe had studied the key influencing factors within the nanopore construction processes by comparing of different types of phospholipid molecule,fluid device,lipid bilayer preparation methods and porins,Through a series of electrochemical detection means,a comprehensive assessment was made on the stability of nanopore system,which ultimately helped to determine a efficient and stable preparation method.The constructed nanopore platform had not only good repeatability,but also high signal-to-noise ratio and resolution,and could achieve single molecule detection level for DNA.3.Single Nucleotide Discrimination by?-Hemolysin NanoporeA well designed 17-mer short probe was respectively annealed with two 57-mer target DNA and forming a complete complementary duplex DNA and a duplex DNA contained a pair of mismatched bases.By analysing of their unzipping processes through the constriction zone of?-HL nanopore channel under a bias voltage,we found the translocation time of the hybridization products was obvious increased than the probe or the template strand.The duplexes only with single base-pair difference have significant differences on unzipping duration.The unzipping duration time of perfect complementary duplex was 430±34 ms,but the duration of single base pair mismatch duplex was only 15±1 ms,which nearly increased by 30 times.Therefore,?-HL nanopore can be used to discriminate single base difference within ssDNA at single molecule level.This simple biosensing strategy can be widely used on detection of SNP sites,disease classification and medical diagnosis.4.Expression and purification of a novel MspA mutant and detection of DNA polymers.By using protein engineering technology,we had replaced three negative charged amino acid residues within the construction zone with uncharged amino acid residues,and three uncharged amino acid residues in the adjacent area of construction zone to positively charged amino acid residues based on the wild-type MspA protein in Escherichia coli.Finally,the new MspA mutant protein was used to detect the DNA polymer of the same chain length,and the results showed that the new type of mutant MspA porin was able to distinguish four different kinds of DNA polymer.However,under the same experimental conditions,the translocation rate of the same DNA homopolymer through the Mut-MspA nanopore decreased nearly 10 times compared with the alpha hemolysin nanopore.In summary,we studied the technical parameter for preparation of nano-electrodes in solid-state nanopores,and subsequently constructs?-hemolysin and MspA mutant biological nanopore systems.The mechanism of translocation and melting behavior of DNA molecules was systematically studied,which provided a theoretical reference for the next step to integrate solid-state nanopores and mutated MspA nanopore to construct solid-biocomposite nanopores.